It is well known that vaccines administered to humans and animals can induce their immune systems to produce antibodies against many pathologic organisms, such as viruses and bacteria. For example, vaccines containing the given antigens, such as hepatitis B surface antigen (HBsAg), human measles virus antigen, malaria antigen, foot-and-mouth disease virus antigen, rabies virus antigen, etc., are important measures to prevent and control such infectious diseases as hepatitis B, measles, malaria, foot-and-mouth disease and rabies in humans or animals. The traditional vaccines are produced from killed or live attenuated pathogens. Recently, several academic and industrial laboratories have begun experimenting with transgenic microorganisms, plants or animals as novel manufacturing systems. However, the applications of these methods are limited greatly for their intrinsic defects which follow: a) some microorganism expression systems lack post-translational modifications, for example, protein glycosylation to eukaryotic proteins. Some insoluble aggregates, for which to be re-dissolved is very difficult, often emerge during fermentation of Escherichia coli. In addition, a huge investment of equipments is often required for the fermentation; b) a high cost is required for culturing animal cells, and what is more serious is that the recombinant proteins produced by transgenic animals may be contaminated with pathogenic viruses from animals, which may be a potential danger to humans.
At present, for example, the hepatitis B vaccine used clinically consists of major protein particles of HBsAg expressed mainly by Saccharomyces cerevisiae. Although the vaccine is effective and plays an important role in controlling transmission of hepatitis B virus, it still has many shortcomings, such as low or no reactivity to some people, immune escape after inoculation, and high price which prevents itself from using widely in the developing countries. Hence, the development of a more effective, and cheaper hepatitis B vaccine is significant for world-widely controlling hepatitis B virus infection.
As compared with other production systems such as microorganism fermentation and transgenic animals, plant as a bioreactor for producing pharmaceutical proteins is safer and cheaper due to the following reasons: firstly, plant viruses do not infect humans, so it is safer; secondly, neither expensive culture materials nor complicated equipments are needed for culture of plant cells; finally, strictly aseptic production conditions and a cold storage can be omitted during distributing.
The biomedical studies on transgenic plants have been made since 1990's. For example, the expression of HBsAg in tobacco was successfully demonstrated in 1992, and later many studies were carried out on some edible plants including tomato, lupine, lettuce, etc. The studies mentioned above have shown that these plants can express human hepatitis B surface antigen protein, but the yield of protein is low. These plants are not often eaten in raw due to seasonal growth thereof. Therefore, it is difficult for them to be adopted widely among the population, especially children in which hepatitis B virus infection is a major health problem.
Hence, it is an objective of the present invention to provide a new and effective transgenic Dunaliella salina bioreactor, which can be used to produce many valuable pharmaceutical proteins including vaccines for humans and/or animals.
It is another objective of the present invention to provide a method for preparing the transgenic Dunaliella Salina bioreactor which is used to produce pharmaceutical proteins, vaccines and hormones suitable for humans, animals and plants.
Unless indicated otherwise, the term “Dunaliella Salina” refers to a kind of organism classified as Chlorophyta; Chlorophyceae; Volvocales; Dunaliellaceae. Dunaliella Salina is, in the shape of pear or ellipse with volume of 50˜1000 μm3, the most halotolerent unicellular eukaryotic organism known. The cell of Dunaliella Salina has two long apical flagella that propel it through the water. The significant difference between Dunaliella Salina and other green unicellular algae is that the cell of Dunaliella Salina lacks a rigid cell wall, and is enclosed by a thin layer of elastic plasma membrane. In the cell, there is the single, large, cup-shaped chloroplast with a pyrenoid. The red eyespot is located at the front end of the cell. Reproduction of Dunaliella Salina thereof includes asexual reproduction in longitudinal split as the main manner and sexual reproduction in isogamy.
The term “transgene”, as used herein, refers to a foreign double-stranded deoxyribonucleic acid (DNA) fragment introduced into Dunaliella Salina as host, which can be either extrachromosomal or integrated into the host genome, and the resulting transgenic Dunaliella Salina can be propagated by normal breeding or not.
The term “foreign target gene” refers to the DNA sequence introduced into the host Dunaliella Salina, which encodes a protein or peptide product.
The term “bioreactor” refers to the transgenic animals, plants or microorganism, which can be used to produce the proteins or peptides encoded by the introduced foreign genes.
The term “selectable marker” refers to the DNA sequence encoding special protein or peptide products, and the host carrying such a sequence can grow, propagate and be screened in a special selective medium.